PROJECT SUMMARY Type 1 diabetes (T1D) remains a clinical problem lacking a robust and specific therapy to address the causative autoimmune attack on the insulin-producing pancreatic beta cells. Our overarching hypothesis is that induction of remission at the hyperglycemic stage will be achieved by targeting key CD8+ T cell specificities, while stabilization of remission, and the establishment of long-term tolerance to beta cells, will be accomplished by fostering regulatory T cells (Treg). We will utilize a series of increasingly humanized murine models, including ones incorporating human MHC molecules, antigens, and T cells, to test this hypothesis. The project represents a collaboration between a T1D immunologist and an expert in T cell co-inhibitory pathways and protein design and production. In Aim 1, induction of remission at the hyperglycemic stage will be accomplished using innovative soluble precision biologics, termed synTacs (artificial immunological synapse for T cell activation), that will enforce activity of the T cell co-inhibitory PD-1 pathway specifically in beta cell-reactive CD8+ T cells, which are important beta cell executioners in both T1D patients and preclinical models. Our uniquely designed synTac reagents covalently link non-exchangeable single-chain peptide-class I MHC and PD-L1 in a manner that recapitulates the proximity, orientation, and overall organization experienced at the immunological synapse. This strategy not only allows for the explicit targeting of disease- relevant T cells, but also greatly reduces the devastating side effects associated with the global immune modulation directed by all biologics currently in the clinic. While inhibition of beta cell-specific CD8+ T cells at the hyperglycemic stage is a promising approach to induce disease remission, we hypothesize that stabilization of remission will require conditions that foster an increased number of effective beta cell-specific Treg. To achieve this goal, in Aim 2 we will take advantage of our experience in targeting antigens to classical dendritic cells (cDC) in vivo using antigen-linked antibodies to cell surface receptors. As cDC1 may be more adept at inducing Treg in the periphery, while cDC2 may be more specialized for the expansion and survival of Treg, we will deliver the critical beta cell antigen proinsulin to cDC1 (via CD205), cDC2 (via DCIR2), or both, to identify the best approach. In Aim 3, we will combine synTac immunotherapy with antigen delivery to cDC to both induce and stabilize remission. Finally, in Aim 4, we will use in vitro and in vivo assays to further investigate the translational potential of our tolerization strategies using human beta cell-specific T cells engineered by lentiviral transduction or obtained from patients. The development of an improved immunodeficient mouse host for the reception of human T cells will enable our combination therapy to be tested in in vivo models incorporating human T cells.